Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. In the past, wars and natural disasters were the main catalysts that promoted epidemics of these ancient afflictions, which are normally transmitted by fleas, lice and ticks. Many of these diseases remain endemic in various regions of the world and therefore pose serious threats to U.S. armed forces troops and civilians who might enter endemic disease zones. A number of these agents have been further weaponized and are widely recognized as being the most significant biothreat agents. Study of disease agents and development of rapid means for their detection take on added importance in light of the use of anthrax for a bioterror attack on the U.S.A. The aim of this proposal is to modify a novel DNA-based methodology we have developed for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of biothreat infectious agents in their natural environments, including intermediate infected hosts, and clinical specimens from humans or infected animals. We plan to use Borrelia burgdorferi, the arthropod-borne etiologic agent of Lyme disease and Yersinia pestis, the etiologic agent of plague, as our principle test agents to work through the systems. We will begin with B. burgdorferi since although it has a complicated life-cycle involving both arthropod and animal intermediates, it is easy to grow and we have extensive experience in working with it in different complex environments including ticks, rodents and human samples. Thus it gives us the opportunity of detecting this pathogen in a variety of complex environments. We also have significant experience with characterizing and identifying subtle changes in the genome of Y. pestis utilizing genomic signature tags. We will use these methods as the foundation of new, high-throughput sequence-based systems to detect zoonotic and/or vector-borne biothreat agents such as Yersinia pestis, Francisella tularensis, Rickettsia ricketsii and other human pathogens such as Ehrlichia and Babesia species. This technology can ultimately be adapted as a sensitive method to detect specific DNA signature sequences from both known and unknown pathogens in a wide variety of complex environments and since it is PCR-based it has the advantage that only minimal quantities of starting material are needed for analysis.